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Joanne K. Tobacman - One of the best experts on this subject based on the ideXlab platform.
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carrageenan induced colonic inflammation is reduced in BCL10 null mice and increased in il 10 deficient mice
Mediators of Inflammation, 2013Co-Authors: Sumit Bhattacharyya, Joanne K. Tobacman, Liquan Xue, Stephan W Morris, Suzanne Devkota, Eugene ChangAbstract:The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μ g/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (BCL10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in BCL10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBα in the colonic epithelial cells were significantly less (P < 0.001) in the carrageenan-treated BCL10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-BCL10, and exogenous IL-10 inhibited only the canonical pathway of NF- κ B activation in cultured colonic cells. These findings demonstrate a BCL10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.
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prolongation of carrageenan induced inflammation in human colonic epithelial cells by activation of an nfκb BCL10 loop
Biochimica et Biophysica Acta, 2012Co-Authors: Alip Borthakur, Pradeep K Dudeja, Sumit Bhattacharyya, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Joanne K. TobacmanAbstract:Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve TLR4- and BCL10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, non-radioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48 hours and increased RelB for 24 hours following withdrawal of carrageenan after 12 hours. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB – BCL10 loop.
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specific effects of BCL10 serine mutations on phosphorylations in canonical and noncanonical pathways of nf κb activation following carrageenan
American Journal of Physiology-gastrointestinal and Liver Physiology, 2011Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. Tobacman, Arivarasu Natarajan Anbazhagan, Shivani KatyalAbstract:To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr559, transforming growth factor-β-activating kinase (TAK) 1 at Thr184, Thr187, and Ser192, and inhibitory factor κBα (IκBα) at Ser32, were examined following mutation of BCL10 at Ser138 and at Ser218. Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser32)-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser138 mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr559 phosphorylation. Hence, the phosphorylation of BCL10 Ser138, but not Ser218, emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser138 or Ser218 mutation inhibited the phosphorylation of TAK1 at Thr184 and at Thr187, but not at Ser192. These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation.
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Lipopolysaccharide-induced activation of NF-κB non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations.
Experimental cell research, 2010Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. TobacmanAbstract:Abstract Background and aims B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-κB (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-κB activation, including nuclear RelB and p52 and phospho- and total NF-κB inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-IκBα of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions LPS induces activation of both canonical and non-canonical pathways of NF-κB in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.
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b cell cll lymphoma 10 BCL10 is required for nf κb production by both canonical and noncanonical pathways and for nf κb inducing kinase nik phosphorylation
Journal of Biological Chemistry, 2010Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. Tobacman, Sangeeta Tyagi, Ravinder K Gill, Mei Ling ChenAbstract:B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ−/− and IKKα−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.
Pradeep K Dudeja - One of the best experts on this subject based on the ideXlab platform.
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prolongation of carrageenan induced inflammation in human colonic epithelial cells by activation of an nfκb BCL10 loop
Biochimica et Biophysica Acta, 2012Co-Authors: Alip Borthakur, Pradeep K Dudeja, Sumit Bhattacharyya, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Joanne K. TobacmanAbstract:Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve TLR4- and BCL10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, non-radioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48 hours and increased RelB for 24 hours following withdrawal of carrageenan after 12 hours. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB – BCL10 loop.
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specific effects of BCL10 serine mutations on phosphorylations in canonical and noncanonical pathways of nf κb activation following carrageenan
American Journal of Physiology-gastrointestinal and Liver Physiology, 2011Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. Tobacman, Arivarasu Natarajan Anbazhagan, Shivani KatyalAbstract:To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr559, transforming growth factor-β-activating kinase (TAK) 1 at Thr184, Thr187, and Ser192, and inhibitory factor κBα (IκBα) at Ser32, were examined following mutation of BCL10 at Ser138 and at Ser218. Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser32)-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser138 mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr559 phosphorylation. Hence, the phosphorylation of BCL10 Ser138, but not Ser218, emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser138 or Ser218 mutation inhibited the phosphorylation of TAK1 at Thr184 and at Thr187, but not at Ser192. These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation.
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carrageenan induced innate immune response is modified by enzymes that hydrolyze distinct galactosidic bonds
Journal of Nutritional Biochemistry, 2010Co-Authors: Haiying Liu, Murielle Jam, Zhenqing Zhang, Pradeep K Dudeja, Gurvan MichelAbstract:The common food additive carrageenan predictably induces intestinal inflammation in animal models. Mechanisms of carrageenan-induced NFκB and IL-8 stimulation include an immune-mediated pathway involving toll-like receptor 4 (TLR4) and B-cell lymphoma/leukemia 10 (BCL10) and a reactive-oxygen species (ROS)-mediated pathway. To determine how the structure of CGN contributes to its initiation of inflammation by these two distinct mechanisms, we treated carrageenans with galactosidases and carrageenases, and determined the impact on IL-8 secretion and BCL10 production. Hydrolysis of carrageenan by the enzyme α-1→(3,6)-galactosidase significantly reduced the increases in IL-8 and BCL10, but other galactosidases tested, including α-1→6-, β-1→4-, and β-1→3,6-galactosidases had no effect. In contrast, specific κ- or ι-carrageenases, which hydrolyze the β-1,4-galactosidic bonds, produced increases in IL-8 and BCL10, attributable to increased exposure of the immunogenic α-1→3-galactosidic epitope of carrageenan to TLR4. These results were consistent with induction of the innate immune response by an interaction of TLR4 with the unusual α-D-Gal-(1→3)-D-Gal epitope that is present in carrageenan. Activation of the ROS-mediated pathway was unaffected by treatment of κ-CGN with either κ-CGNase (3 mg/L), α-1→(3,6)-galactosidase (20 mU/ml), or these enzymes in combination, indicating that the changes in IL-8 production were attributable to effects on the TLR4-BCL10-mediated innate immune pathway of induction of inflammation. These findings provide new information about the specificity of the carbohydrate-protein interaction between carrageenan and TLR4 and may help to devise treatments that modify the immune reactivity induced by carbohydrate antigens.
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Lipopolysaccharide-induced activation of NF-κB non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations.
Experimental cell research, 2010Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. TobacmanAbstract:Abstract Background and aims B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-κB (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-κB activation, including nuclear RelB and p52 and phospho- and total NF-κB inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-IκBα of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions LPS induces activation of both canonical and non-canonical pathways of NF-κB in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.
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b cell cll lymphoma 10 BCL10 is required for nf κb production by both canonical and noncanonical pathways and for nf κb inducing kinase nik phosphorylation
Journal of Biological Chemistry, 2010Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. Tobacman, Sangeeta Tyagi, Ravinder K Gill, Mei Ling ChenAbstract:B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ−/− and IKKα−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.
Sumit Bhattacharyya - One of the best experts on this subject based on the ideXlab platform.
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carrageenan induced colonic inflammation is reduced in BCL10 null mice and increased in il 10 deficient mice
Mediators of Inflammation, 2013Co-Authors: Sumit Bhattacharyya, Joanne K. Tobacman, Liquan Xue, Stephan W Morris, Suzanne Devkota, Eugene ChangAbstract:The common food additive carrageenan is a known activator of inflammation in mammalian tissues and stimulates both the canonical and noncanonical pathways of NF-κB activation. Exposure to low concentrations of carrageenan (10 μ g/mL in the water supply) has produced glucose intolerance, insulin resistance, and impaired insulin signaling in C57BL/6 mice. B-cell leukemia/lymphoma 10 (BCL10) is a mediator of inflammatory signals from Toll-like receptor (TLR) 4 in myeloid and epithelial cells. Since the TLR4 signaling pathway is activated in diabetes and by carrageenan, we addressed systemic and intestinal inflammatory responses following carrageenan exposure in BCL10 wild type, heterozygous, and null mice. Fecal calprotectin and circulating keratinocyte chemokine (KC), nuclear RelA and RelB, phospho(Thr559)-NF-κB-inducing kinase (NIK), and phospho(Ser36)-IκBα in the colonic epithelial cells were significantly less (P < 0.001) in the carrageenan-treated BCL10 null mice than in controls. IL-10-deficient mice exposed to carrageenan in a germ-free environment showed an increase in activation of the canonical pathway of NF-κB (RelA) activation, but without increase in RelB or phospho-BCL10, and exogenous IL-10 inhibited only the canonical pathway of NF- κ B activation in cultured colonic cells. These findings demonstrate a BCL10 requirement for maximum development of carrageenan-induced inflammation and lack of complete suppression by IL-10 of carrageenan-induced inflammation.
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prolongation of carrageenan induced inflammation in human colonic epithelial cells by activation of an nfκb BCL10 loop
Biochimica et Biophysica Acta, 2012Co-Authors: Alip Borthakur, Pradeep K Dudeja, Sumit Bhattacharyya, Arivarasu Natarajan Anbazhagan, Anoop Kumar, Joanne K. TobacmanAbstract:Carrageenan, a sulfated polysaccharide that is widely used as a food additive, induces inflammatory responses in animal models and human cells. The carrageenan-induced inflammatory cascades involve TLR4- and BCL10-dependent activation of NF-κB, leading to increased IL-8 production. Translocations involving BCL10 in the mucosa-associated lymphoid tissue (MALT) lymphomas are associated with constitutive activation of NF-κB. This report presents a mechanism by which carrageenan exposure leads to prolonged activation of both BCL10 and NF-κB in human colonic epithelial cells. Study findings demonstrate that nuclear RelA and RelB bind to an NF-κB binding motif in the BCL10 promoter in human colonic epithelial NCM460 and HT-29 cells. In vitro oligonucleotide binding assay, non-radioactive gel shift assay, and chromatin immunoprecipitation (ChIP) indicate binding of RelA and RelB to the BCL10 promoter. Prolonged inflammation follows activation of the BCL10-NFκB inflammatory loop in response to carrageenan, shown by increased BCL10, RelA, and IL-8 for 36 to 48 hours and increased RelB for 24 hours following withdrawal of carrageenan after 12 hours. In contrast, exposure to dextran sulfate sodium, which does not cause inflammation through TLR4 and BCL10 in the colonic epithelial cells, did not provoke prolonged activation of inflammation. The carrageenan-enhanced BCL10 promoter activity was blocked by caffeic acid phenethyl ester (CAPE) and MB-132 which inhibit NF-κB activation. These results indicate that NF-κB binding to the BCL10 promoter can lead to prolonged activation of the carrageenan-induced inflammatory cascade by a transcriptional mechanism involving an NF-κB – BCL10 loop.
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specific effects of BCL10 serine mutations on phosphorylations in canonical and noncanonical pathways of nf κb activation following carrageenan
American Journal of Physiology-gastrointestinal and Liver Physiology, 2011Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. Tobacman, Arivarasu Natarajan Anbazhagan, Shivani KatyalAbstract:To determine the impact of B cell leukemia/lymphoma (BCL) 10 on the phosphorylation of crucial mediators in NF-κB-mediated inflammatory pathways, human colonic epithelial cells were exposed to carrageenan (CGN), a sulfated polysaccharide commonly used as a food additive and known to induce NF-κB nuclear translocation by both canonical and noncanonical pathways. Phosphorylations of intermediates in inflammatory cascades, including NF-κB-inducing kinase (NIK) at Thr559, transforming growth factor-β-activating kinase (TAK) 1 at Thr184, Thr187, and Ser192, and inhibitory factor κBα (IκBα) at Ser32, were examined following mutation of BCL10 at Ser138 and at Ser218. Specific phosphoantibodies were used for detection by enzyme-linked immunosorbent assay, immunoblot, and confocal microscopy of differences in phosphorylation following transfection by mutated BCL10. Both mutations demonstrated dominant-negative effects, with inhibition of phospho(Ser32)-IκBα to less than control levels. Both of the BCL10 mutations reduced the CGN-induced increases in nuclear RelA and p50, but only the Ser138 mutation inhibited the CGN-induced increases in nuclear RelB and p52 and in NIK Thr559 phosphorylation. Hence, the phosphorylation of BCL10 Ser138, but not Ser218, emerged as a critical event in activation of the noncanonical pathway of NF-κB activation. Either BCL10 Ser138 or Ser218 mutation inhibited the phosphorylation of TAK1 at Thr184 and at Thr187, but not at Ser192. These findings indicate that BCL10 phosphorylations act upstream of phosphorylations of NIK, TAK1, and IκBα and differentially affect the canonical and noncanonical pathways of NF-κB activation.
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Lipopolysaccharide-induced activation of NF-κB non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations.
Experimental cell research, 2010Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. TobacmanAbstract:Abstract Background and aims B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-κB (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease. Methods Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-κB activation, including nuclear RelB and p52 and phospho- and total NF-κB inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined. Results By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-IκBα of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images. Conclusions LPS induces activation of both canonical and non-canonical pathways of NF-κB in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.
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b cell cll lymphoma 10 BCL10 is required for nf κb production by both canonical and noncanonical pathways and for nf κb inducing kinase nik phosphorylation
Journal of Biological Chemistry, 2010Co-Authors: Sumit Bhattacharyya, Pradeep K Dudeja, Alip Borthakur, Joanne K. Tobacman, Sangeeta Tyagi, Ravinder K Gill, Mei Ling ChenAbstract:B-cell CLL/lymphoma 10 (BCL10), the caspase recruitment domain (CARD)-containing protein involved in the etiology of the mucosa-associated lymphoid tissue (MALT) lymphomas, has been implicated in inflammatory processes in epithelial cells, as well as in immune cells. Experiments in this report indicate that BCL10 is required for activation of nuclear factor (NF)-κB by both canonical and noncanonical pathways, following stimulation by the sulfated polysaccharide carrageenan (CGN). In wild type and IκB-kinase (IKK)α−/− mouse embryonic fibroblasts, increases in phospho-IκBα, nuclear NF-κB p65 (RelA) and p50, and KC, the mouse analog of human interleukin-8, were markedly reduced by silencing BCL10 or by exposure to the free radical scavenger Tempol. In IKKβ−/− cells, BCL10 silencing, but not Tempol, reduced the CGN-induced increases in KC, phospho-NF-κB-inducing kinase (NIK), cytoplasmic NF-κB p100, and nuclear NF-κB p52 and RelB, suggesting a BCL10 requirement for activation of the noncanonical pathway. In NCM460 cells, derived from normal, human colonic epithelium, the CGN-induced increases in NF-κB family members, p65, p50, p52, and RelB, were inhibited by BCL10 silencing. Although enzyme-linked immunosorbent assay and confocal images demonstrated no change in total NIK following CGN, increases in phospho-NIK in the wild type, IKKβ−/− and IKKα−/− cells were inhibited by silencing BCL10. These findings indicate an upstream signaling role for BCL10, in addition to its effects on IKKγ, the regulatory component of the IKK signalosome, and a requirement for BCL10 in both canonical and noncanonical pathways of NF-κB activation. Also, the commonly used food additive carrageenan can be added to the short list of known activators of both pathways.
Jürgen Ruland - One of the best experts on this subject based on the ideXlab platform.
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BCL10 links saturated fat overnutrition with hepatocellular nf kb activation and insulin resistance
Cell Reports, 2012Co-Authors: Matthew Van Beek, Ingrid J Apel, Katherine Oraveczwilson, Phillip C Delekta, Shufang Gu, Xiangquan Li, Katy S Konkle, Yongjia Feng, Daniel H Teitelbaum, Jürgen RulandAbstract:Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. With high-fat feeding, FFAs activate NF-kB in target tissues, initiating negative crosstalk with insulin signaling. However, the mechanisms underlying FFA-dependent NF-kB activation remain unclear. Here, we demonstrate that the saturated FA, palmitate, requires BCL10 for NF-kB activation in hepatocytes. Uptake of palmitate, metabolism to diacylglycerol, and subsequent activation of protein kinase C (PKC) appear to mechanistically link palmitate with BCL10, known as a central component of a signaling complex that, along with CARMA3 and MALT1, activates NF-kB downstream of selected cell surface receptors. Consequently, BCL10-deficient mice are protected from hepatic NF-kB activation and insulin resistance following brief high-fat diet, suggesting that BCL10 plays a major role in the metabolic consequences of acute overnutrition. Surprisingly, while CARMA3 also participates in the palmitate response, MALT1 is completely dispensable, thereby revealing an apparent nonclassical role for BCL10 in NF-kB signaling.
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inhibition of malt1 protease activity is selectively toxic for activated b cell like diffuse large b cell lymphoma cells
Journal of Experimental Medicine, 2009Co-Authors: Uta Ferch, Andreas Gewies, Michael Düwel, Bernhard Kloo, Daniel Krappmann, Vera Pfänder, Christian Peschel, Jürgen RulandAbstract:Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell–like (ABC) subtype of DLBCL is characterized by constitutive NF-κB activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11–BCL10–MALT1 (CBM) complexes couple upstream events to IκB kinase (IKK)/NF-κB activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-κB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell–like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-κB activity, and decreases the expression of NF-κB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.
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BCL10 and malt1 control lysophosphatidic acid induced nf kappab activation and cytokine production
Proceedings of the National Academy of Sciences of the United States of America, 2007Co-Authors: Stefanie Klemm, Tak W Mak, Christian Peschel, Stephanie Zimmermann, Jürgen RulandAbstract:Lysophosphatidic acid (LPA) is a potent bioactive phospholipid that stimulates a variety of cellular responses by acting on cognate G protein-coupled receptors (GPCRs). There is increasing evidence that LPA signaling reprograms gene expression, but the GPCR-induced pathways connecting LPA receptor stimulation to downstream transcription factors are not well characterized. Here, we identify the adapter proteins BCL10 and Malt1 as essential mediators of LPA-induced NF-κB activation. Both proteins were previously known to activate NF-κB in response to antigen receptor ligation on lymphocytes, but their functions in nonimmune cells are still largely undefined. By using murine embryonic fibroblasts from BCL10- or Malt1-deficient mice as a genetic model, we report that BCL10 and Malt1 are critically required for the degradation of IκB-α and the subsequent NF-κB induction in response to LPA stimulation. BCL10 and Malt1 cooperate with PKCs selectively for LPA-induced NF-κB activation but are dispensable for the activation of the Jnk, p38, Erk MAP kinase, and Akt signaling pathways. In a biological readout, we demonstrate that LPA-induced IL-6 production is abolished in the absence of BCL10. Thus, our results identify a NF-κB-inducing signaling pathway downstream of GPCRs and reveal previously unrecognized functions for BCL10/Malt1 signaling in nonimmune cells.
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essential role for iκb kinase β in remodeling carma1 BCL10 malt1 complexes upon t cell activation
Molecular Cell, 2006Co-Authors: Elmar Wegener, Uta Ferch, Jürgen Ruland, Andrea Oeckinghaus, Nikoletta Papadopoulou, Liron Lavitas, Marc Schmidtsupprian, Tak W Mak, Vigo HeissmeyerAbstract:T cell receptor (TCR) signaling to IkappaB kinase (IKK)/NF-kappaB is controlled by PKCtheta-dependent activation of the Carma1, BCL10, and Malt1 (CBM) complex. Antigen-induced phosphorylation of BCL10 has been reported, but its physiological function is unknown. Here we show that the putative downstream kinase IKKbeta is required for initial CBM complex formation. Further, upon engagement of IKKbeta/Malt1/BCL10 with Carma1, IKKbeta phosphorylates BCL10 in the C terminus and thereby interferes with BCL10/Malt1 association and BCL10-mediated IKKgamma ubiquitination. Mutation of the IKKbeta phosphorylation sites on BCL10 enhances expression of NF-kappaB target genes IL-2 and TNFalpha after activation of primary T cells. Thus, our data provide evidence that IKKbeta serves a dual role upstream of its classical substrates, the IkappaB proteins. While being essential for triggering initial CBM complex formation, IKKbeta-dependent phosphorylation of BCL10 exhibits a negative regulatory role in T cell activation.
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Differential Requirement for Malt1 in T and B Cell Antigen Receptor Signaling
Immunity, 2003Co-Authors: Jürgen Ruland, Gordon S Duncan, Andrew WakehamAbstract:Abstract The translocation t(11;18)(q21;q21) involving MALT1 is the most common chromosomal abnormality in lymphomas of mucosa-associated lymphoid tissue. Although the paracaspase MALT1 can bind to BCL10, the physiological function of MALT1 is unknown. Using mouse models, we show that Malt1 is essential for T cell activation, proliferation, and IL-2 production in response to TCR ligation and strictly required for signal-specific NF-κB activation induced by the TCR but not TNF-α or IL-1 signaling. Malt1 operates downstream of BCL10, controls the catalytic activity of the canonical IKK complex, and regulates the signaling of Jnk and p38 MAP kinases. In contrast to BCL10 disruption, however, inactivation of Malt1 has only mild effects on B cell activation and does not cause defects during neurodevelopment. Thus, Malt1 is an essential regulator of BCL10 signaling that is differentially required depending on cellular context.
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BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation.
Frontiers in immunology, 2018Co-Authors: Susanne Kurz, Katja Lammens, Florian Schlauderer, Torben Gehring, Simone Woods, Simon Widmann, Daniel KrappmannAbstract:Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.
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Molecular architecture and regulation of BCL10-MALT1 filaments
Nature Communications, 2018Co-Authors: Florian Schlauderer, Daniel Krappmann, Thomas Seeholzer, Torben Gehring, Irina Gutsche, Ambroise Desfosses, Mike Strauss, Karl-peter Hopfner, Katja LammensAbstract:The CARD11-BCL10-MALT1 (CBM) complex triggers the adaptive immune response in lymphocytes and lymphoma cells. CARD11/CARMA1 acts as a molecular seed inducing BCL10 filaments, but the integration of MALT1 and the assembly of a functional CBM complex has remained elusive. Using cryo-EM we solved the helical structure of the BCL10-MALT1 filament. The structural model of the filament core solved at 4.9 A resolution identified the interface between the N-terminal MALT1 DD and the BCL10 caspase recruitment domain. The C-terminal MALT1 Ig and paracaspase domains protrude from this core to orchestrate binding of mediators and substrates at the filament periphery. Mutagenesis studies support the importance of the identified BCL10-MALT1 interface for CBM complex assembly, MALT1 protease activation and NF-κB signaling in Jurkat and primary CD4 T-cells. Collectively, we present a model for the assembly and architecture of the CBM signaling complex and how it functions as a signaling hub in T-lymphocytes.
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BCL10 bridging cards to immune activation
Frontiers in Immunology, 2018Co-Authors: Torben Gehring, Thomas Seeholzer, Daniel KrappmannAbstract:Since the B-cell lymphoma/leukemia 10 (BCL10) protein was first described in 1999, numerous studies have elucidated its key functions in channeling adaptive and innate immune signaling downstream of CARMA/caspase-recruitment domain (CARD) scaffold proteins. While T and B cell antigen receptor (TCR/BCR) signaling induces the recruitment of BCL10 bound to mucosa-associated lymphoid tissue (MALT)1 to the lymphocyte-specific CARMA1/CARD11-BCL10-MALT1 (CBM-1) signalosome, alternative CBM complexes utilize different CARMA/CARD scaffolds in distinct innate or inflammatory pathways. BCL10 constitutes the smallest subunit in all CBM signalosomes, containing a 233 amino acid coding for N-terminal CARD as well as a C-terminal Ser/Thr-rich region. BCL10 forms filaments, thereby aggregating into higher-order clusters that mediate and amplify stimulation-induced signals, ultimately leading to MALT1 protease activation and canonical NF-κB and JNK signaling. BCL10 additionally undergoes extensive post-translational regulation involving phosphorylation, ubiquitination, MALT1-catalyzed cleavage, and degradation. Through these feedback and feed-forward events, BCL10 integrates positive and negative regulatory processes that govern the function as well as the dynamic assembly, disassembly, and destruction of CBM complexes. Thus, BCL10 is a critical regulator for activation as well as termination of immune cell signaling, revealing that its role extends far beyond that of a mere linking factor in CBM complexes.
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Data_Sheet_1_BCL10-CARD11 Fusion Mimics an Active CARD11 Seed That Triggers Constitutive BCL10 Oligomerization and Lymphocyte Activation.PDF
2018Co-Authors: Thomas Seeholzer, Katja Lammens, Florian Schlauderer, Torben Gehring, Susanne Kurz, Simone Woods, Simon Widmann, Daniel KrappmannAbstract:Assembly of the CARD11/CARMA1-BCL10-MALT1 (CBM) signaling complex upon T or B cell antigen receptor (TCR or BCR) engagement drives lymphocyte activation. Recruitment of pre-assembled BCL10-MALT1 complexes to CARD11 fosters activation of the MALT1 protease and canonical NF-κB signaling. Structural data and in vitro assays have suggested that CARD11 acts as a seed that nucleates the assembly of BCL10 filaments, but the relevance of these findings for CBM complex assembly in cells remains unresolved. To uncouple cellular CARD11 recruitment of BCL10 and BCL10 filament assembly, we generated a BCL10-CARD11 fusion protein that links the C-terminus of BCL10 to the N-terminus of CARD11. When stably expressed in CARD11 KO Jurkat T cells, the BCL10-CARD11 fusion induced constitutive MALT1 activation. Furthermore, in CARD11 KO BJAB B cells, BCL10-CARD11 promoted constitutive NF-κB activation to a similar extent as CARD11 containing oncogenic driver mutations. Using structure-guided destructive mutations in the CARD11-BCL10 (CARD11 R35A) or BCL10-BCL10 (BCL10 R42E) interfaces, we demonstrate that chronic activation by the BCL10-CARD11 fusion protein was independent of the CARD11 CARD. However, activation strictly relied upon the ability of the BCL10 CARD to form oligomers. Thus, by combining distinct CARD mutations in the context of constitutively active BCL10-CARD11 fusion proteins, we provide evidence that BCL10-MALT1 recruitment to CARD11 and BCL10 oligomerization are interconnected processes, which bridge the CARD11 seed to downstream pathways in lymphocytes.
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inhibition of malt1 protease activity is selectively toxic for activated b cell like diffuse large b cell lymphoma cells
Journal of Experimental Medicine, 2009Co-Authors: Uta Ferch, Andreas Gewies, Michael Düwel, Bernhard Kloo, Daniel Krappmann, Vera Pfänder, Christian Peschel, Jürgen RulandAbstract:Diffuse large B cell lymphoma (DLBCL) is the most common type of lymphoma in humans. The aggressive activated B cell–like (ABC) subtype of DLBCL is characterized by constitutive NF-κB activity and requires signals from CARD11, BCL10, and the paracaspase MALT1 for survival. CARD11, BCL10, and MALT1 are scaffold proteins that normally associate upon antigen receptor ligation. Signal-induced CARD11–BCL10–MALT1 (CBM) complexes couple upstream events to IκB kinase (IKK)/NF-κB activation. MALT1 also possesses a recently recognized proteolytic activity that cleaves and inactivates the negative NF-κB regulator A20 and BCL10 upon antigen receptor ligation. Yet, the relevance of MALT1 proteolytic activity for malignant cell growth is unknown. Here, we demonstrate preassembled CBM complexes and constitutive proteolysis of the two known MALT1 substrates in ABC-DLBCL, but not in germinal center B cell–like (GCB) DLBCL. ABC-DLBCL cell treatment with a MALT1 protease inhibitor blocks A20 and BCL10 cleavage, reduces NF-κB activity, and decreases the expression of NF-κB targets genes. Finally, MALT1 paracaspase inhibition results in death and growth retardation selectively in ABC-DLBCL cells. Thus, our results indicate a growth-promoting role for MALT1 paracaspase activity in ABC-DLBCL and suggest that a pharmacological MALT1 protease inhibition could be a promising approach for lymphoma treatment.